Chronic heavy ethanol consumption has major adverse effects on the mammalian liver, namely the induction of hepatocellular damage and impairment of regeneration following injury. For a medication development project focusing on alcohol liver diseases, it is essential to understand, at the molecular level, potential alterations in cellular signal transduction pathways that adversely affect the hepatocyte proliferative response, as well as those survival mechanisms critical for liver regeneration. The objective of this STTR Phase I and II fast track application is to develop a naturally occurring agent, namely Silybin (NPI-MK-001), as a hepatoprotective agent for alcohol liver problems. Based on our preliminary studies, NPI-MK-001 is the bioactive component isolated from the seed of Milk thistle (Silybum marianum), the best-studied medicinal plant for the treatment of liver disease. We plan to study the cytoprotective effect of NPIMK-001 on liver injury produced by chronic ethanol consumption in rat hepatocyte culture. We will also evaluate the therapeutic potential of NPI-MK-001 on the hepatic repair process in vivo by analyzing effects on specific growth factor signal transduction cascades associated with hepatocyte DNA synthesis. Our specific aims are: Aim #1 - Determine the in vitro cytoprotective effects of NPI-MK-001 on hepatocyte injury caused by chronic ethanol feeding+ hypoxia; Aim #2 - Explore the in vivo action of NPI-MK-001 on the IRS-1 mediated growth factor signal transduction pathway associated with liver repair in chronic ethanol-fed rats and IRS-1 transgenic mice. In Phase I, we will focus on the standardization of NPI-MK-001, a prerequisite for in vitro and in vivo evaluation. We propose to (1) conduct field inspection of the collection, processing, extraction, and contamination control (insecticides, herbicides, and chemical fertilizers) of the seed of Milk Thistle; (2) optimize the analytical HPLC conditions for qualitative and quantitative analysis of NPI-MK-001; (3) isolate and purify a sufficient quantity of NPI-MK-001 and its diasteric isomers (Silybin A and B) for chemical markers, and for in vitro and in vivo evaluation; and (4) develop a cost-effective separation procedure for large scale purification of NPI-MK-001. In Phase II, we propose to: (1) evaluate relative potency of NPI-MK-001and its diasteric isomers (Silybin A and Silybin B) by in vitro and in vivo models; (2) investigate the mechanism of action of NPI-MK-001 as specified in Aims 1 and 2; (3) Synthesize radio-labeled NPIMK-001 and assess the bioavailability and pharmcokinetics; (4) standardize NPI-MK-001 and corresponding placebo according to FDA guidelines; (5) obtain data on mutagenesis and acute and chronic toxicity; (6) conduct stability testing of the standardized NPI-MK-001; and (7) establish protocols for the capsulation of standardized NPI-MK-001 and placebo under GMP conditions. Natural Pharmacia International, Inc, an established natural products laboratory, has assembled an excellent consortial team to assure that the quality and purity of NPI-MK-001would meet the stringent FDA requirements. The key to the success of this STTR program is not merely the exceptional experience of the team members in carry out the proposed studies in Phases I and II, but also the outstanding business development plan which supports the further development of NPI-MK-001 including filing of a DMF and an IND in Phase III.